The dynamic process of membrane trafficking within eukaryotic cells involves numerous specialized protein complexes and lipid domains. One particularly intriguing set of proteins is the dynamin family of mechanochemical enzymes; a family of large GTPases potentially involved in nearly all cellular membrane stabilization and fission events. We are interested in examining the dynamic structural properties of these proteins, derived from their mechanochemical properties, and correlate them to their cellular function. Dynamin itself is essential for receptor mediated endocytosis, caveolae internalization and trafficking to and from the Golgi. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue to the shibire gene product in Drosophila. Since then human dynamin mutants overexpressed in mammalian cells were found to effectively block clathrin-mediated endocytosis. We have previously shown that purified dynamin readily assembles into rings and spirals, and assembles onto liposomes forming dynamin-lipid tubes that constrict upon GTP hydrolysis. This evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. To further explore the dynamics of dynamin during GTP hydrolysis we applied the novel technique of time-resolved cryo-electron microscopy. We observed that immediately upon GTP addition (within seconds) dynamin constricts the underlying lipid bilayer in a concerted action and excess lipid bulges out at focal points along the constricted tubes. Following constriction, dynamin falls off the lipid bilayer suggesting the dynamin-dynamin interactions are unstable in the constricted state. We are currently exploring different dynamin mutants, lipid, nucleotide and temperature conditions to mimic the in vivo environment. In 2001, we calculated the first three-dimensional map of dynamin in the constricted state using cryo-electron microscopy and helical reconstruction methods at a resolution of 20 Angstroms. The map was determined using a dynamin mutant lacking the proline rich C-terminus (delta-PRD) in the presence of a GTP analogue. The 3D map consists of a repeating T structure (dimer) along the tube axis, which can be divided into three distinct densities called head, stalk and leg. Based on previous biochemical results and the docking of X-ray crystal structures into our map, we predict that the GTPase domain is located in the head and the PH domain is located in the leg. This leaves the middle domain and GTPase effector domain (GED) most likely located in the stalk. The positioning of GED within the stalk fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. The constriction observed by GTP addition causes a decrease in both radial diameter and axial repeat. Based on our 3D map, an interaction between GED and a GTPase domain from a neighboring dimer could lead to both a radial and axial constriction. In 2004 we solved the structure of dynamin in the non-constricted state using single particle reconstruction methods. Comparison of the 3D maps in the constricted and non-constricted states shows a large conformational change in the GED domain and suggests the GED interacts in trans with the GTPase domain to cause constriction. Currently we are pursuing a high-resolution map, less than10 Angstroms, of full-length dynamin in the constricted state. Additional dynamin family members have been implicated in numerous fundamental cellular processes, including other membrane fission events, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures (i.e. rings and spirals) is a common characteristic and, for the majority, essential for their function. While they are continually being implicated in diverse functions of the cell, we would like to know if a common mechanism of action exists. To address this problem, we are currently examining the dynamin family member Drp1 (Dnm1), a dynamin related protein involved in mitochondria fission. We have shown that Dnm1 assembles into large spirals, 100 nm in diameter compared to the 50 nm dynamin spirals. The large Dnm1 spirals remarkably resemble mitochondria constriction sites observed in yeast. These results suggest that the structural properties of dynamin family members are uniquely tailored to fit their function. In addition, the binding of Dnm1 to its partner Mdv1 depends on the assembly state of Dnm1: Mdv1 readily binds Dnm1 in the spiral state only. We are currently examining the effects of GTP on Dnm1-lipid tubes to determine if this protein is capable of membrane constriction. In the future we plan to examine several other dynamin family members including Opa1, a protein most abundant in retinal cells with mutations related to optic atrophy type 1 (OPA1), an inherited optical neuropathy disease. It is unclear how this dynamin homolog can aid in membrane fusion, since most dynamin family are believed to be involved in fission events. Our structural studies of Dnm1 and Opa1p using electron microscopy, light scattering and biochemical methods will provide valuable details of the mechanism required to balance the fission and fusion events in the outer and inner mitochondrial membranes.